The broad aim of this project is to understand at the cellular and molecular level the mechanisms by which surface membrane receptors for hormones, neurotransmitters and growth factors modify cellular responses through mobilization of cellular Ca2+. An early event in the action of receptors of this class is the hydrolysis of a membrane lipid, phosphatidylinositol 4,5-bisphosphate to a Ca2+-mobilizing second messengers, which releases Ca2+ from an intracellular organelle. The general approach in this project is to combine HPLC measurements of the formation and metabolism of inositol phosphates with real time measurements of cytosolic Ca2+ using intracellular fluorescent Ca2+ indicators. Our previous work has shown that a mechanism exists for signalling the entry of Ca2+ into cells following the depletion of Ca2+ from an intracellular organelle through the action of IP3. We are currently attempting to understand the molecular mechanism underlying this process utilizing as a model the Ca2+-mobilizing actions of the tumor promoter, thapsigargin, which activates this process while bypassing surface receptors and the generation of IP3. We are also trying to characterize the Ca2+ entry channel using PCR-cloning techniques. We have discovered a novel technique for imaging intracellular Ca2+ pools in living cells, and in collaboration with investigators in the Laboratory of Molecular Biophysics, have developed a novel calcium indicator to facilitate this kind of Ca2+ imaging. Since the Ca2+ signalling system is centrally involved in the regulation of cellular growth under normal and pathological (neoplastic) conditions, these studies may provide novel perspectives on the pharmacological regulation and arrest of these processes. In addition, Ca2+ is believed to play a central role in mechanisms of chemically-induced cell injury, and thus these studies should provide insights into the mechanisms underlying the pathophysiological consequences of exposure to toxins and other environmental agents.